In 2008, the FACS Core Lab provided service to 26 NCI labs or branches and 2 institutes. These labs, with the numbers of principal investigators using the FACS Core in parentheses, are: CCBB (6), Dermatology Branch (2), Genetics Branch (3), LBMB (3), LCB (2), LCBG (8), LCMB (5), LCO (4), LCP (1), LEC (1), LHC (3), LICB (3), LM (5), LMB (5), LMP (5), LPG (1), LRBGE (1), LTIB (3), MBTL (3), Medical Oncology Branch (3), Neuro-Oncology Branch (1), OD (1), Pediatric Oncology Branch (1), Radiation Oncology Branch (1), Surgery Branch (2), NCI-Frederick (1), NICHD (1), and NINDS (1). From these labs, 205 scientists have used the FACS Core in FY2008 and of these, 101 were new to the Core lab this past year. Identifying and studying cancer stem cells is one of the major research areas of the FACS Core Lab users. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, LCBG, LM, and Surgery Branch are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic, and lung carcinomas. The LSRII flow cytometer, the FACS Vantage cell sorter, and the new special order FACS Aria, because each is equipped with a UV laser, are frequently used for these assays. This has contributed to a 40% increase in LSRII usage over the previous year and a 45% increase in sorting hours over last year. Both increases were made possible by the addition of a full-time biologist to the FACS Core. Many NCI labs use the FACS Core to study regulation of cell cycle and apoptotic pathways since dysregulations in each may be crucial to development of tumors. Tumorigenic substances and anti-cancer agents both act upon these pathways. The 2 FACSCaliburs are suited to most of these assays and there was a 10% increase in Calibur usage over last year. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of the labs using the FACS Core. The FACS Core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with any of the green, yellow, blue, red, and UV fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have then been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of luciferase. These cells have then been used to establish tumors in mice and to image metastasis. In addition to the 3 co-authored publications, 12 additional publications this year have included work done in the FACS Core and/or acknowledgments of FACS Core participation.